The regulation of protein synthesis at the level of translation initiation plays a key role in the control of cell growth, proliferation, and apoptosis. Translation, the mRNA-directed synthesis of proteins, occurs in three distinct steps: initiation, elongation and termination. Translation initiation is a complex process in which the two ribosomal subunits and methionyl tRNA (met-tRNAi) assemble on a properly aligned mRNA to commence chain elongation at the AUG initiation codon. The interaction between the initiation factors eIF4E and eIF4G is a major component of this process. eIF4E binds the 7-methylguanosine cap structure found at the 5′ ends of most messenger RNAs. Its binding partner eIF4G, a scaffold protein, provides a docking site for other initiation factors, including the RNA helicase eIF4A. Collectively, eIF4E, eIF4G, and eIF4A form a ternary complex referred to as eIF4F. Once assembled, this complex recruits the 40S ribosomal subunit to the 5′ end of the mRNA molecule as a result of the interaction of eIF3 with eIF4G, followed by scanning of the 40S subunit to the initiation codon where it joins with the 60S subunit. This process is facilitated by eIF4A, with the requirement for its helicase activity being directly proportional to the amount of secondary structure in the 5′ UTR that must be melted for scanning to occur.
Translation initiation is a critical step in the regulation of cell growth because the expression of most oncogenes and cell growth regulatory proteins is translationally regulated. Biosynthesis of many growth-promoting proteins is suppressed on the translation-initiation level, and several forms of cancer exhibit an out-of-balance translation initiation machinery. Although inhibitors of translation exist, most, if not all, act nonspecifically on all translation.
Many types of tumor cells are characterized by aberrant protein translation initiation mechanisms, e.g., association or binding of certain translation initiation factors. For example, the interaction of the cap-binding protein eIF4E with the mRNA cap, the scaffold protein eIF4G, and the regulatory 4E-BPs, are involved in cell transformation. Small-molecule inhibitors of the eIF4E/eIF4G interaction have been identified and found to possess anti-tumor activity.
Recruitment of the capped 5′ end of an mRNA to the small ribosomal subunit is thought to be the major rate-limiting step in eukaryotic translation initiation. This process is tightly regulated and requires the stepwise assembly of a large multiprotein complex centered on the trimeric complex eIF4F, comprised of the translation initiation factors eIF4E, eIF4G, and eIF4A. Cap-bound eIF4F recruits the 40S ribosomal subunit through the interaction of eIF3 with eIF4G, which initiates scanning to the initiation codon where it joins with the 60S subunit. This process is facilitated by eIF4A, with the requirement for its helicase activity directly proportional to the amount of secondary structure in the 5′ UTR that must be melted for scanning to occur. All eIF4G proteins bind eIF4E through a motif of sequence Y(X)4LΦ, where X is variable and Φ is hydrophobic. This motif forms a helical peptide structure, which binds a conserved surface of hydrophobic residues on the dorsal side of eIF4E.
Cellular mRNAs differ greatly in their requirement for eIF4F for efficient translation and in the composition of the 5′ UTR. The majority of growth and proliferation related proteins are encoded by “weak” mRNAs containing long highly structured 5′ UTRs which have lower translational efficiency than “strong” mRNAs, which contain relatively short and unstructured 5′ UTRs. Translation of weak mRNAs is highly eIF4F dependent and is preferentially enhanced when the level of eIF4F complex is increased by eIF4E overexpression. The amount of eIF4E available for complex formation is controlled by a class of small proteins termed 4E-BPs which contain the Y(X)4LΦ motif and bind to the same surface as eIF4G. In response to stimuli such as nutrients and growth factors 4E-BPs undergo a set of hierarchical phosphorylation events. Hyperphosphorylated forms of 4E-BPs bind eIF4E much more weakly than hypophosphorylated forms, and thus 4E-BP phosphorylation acts as a switch to up-regulate the level of eIF4F and cap-dependent translation.
Misregulation of cap-dependent translation due to overexpression of eIF4E and the other components of the eIF4F complex is thought to play an important role in the development of many forms of cancer. In cultured mammalian cells overexpression of eIF4E or eIF4G induces malignant transformation while overexpression of 4E-BP1 partially reverses transformation by eIF4E. In addition, etopic expression of nonphosphorylatable forms of 4E-BP1 can inhibit proliferation and/or induce apoptosis in cancer cell lines. Inhibition of the eIF4F complex is useful for cancer therapy. See PCT/US2006/002093 hereby incorporated by reference in its entirety.
The disruption of proper translational regulation by elevated levels of eIF4F complexes is an important factor in carcinogenesis. A wide variety of tumors have been found to have abnormally elevated eIF4E levels, and eIF4G is amplified in some lung cancers. The overexpression of eIF4E in cultured cells can cause them to exhibit a malignant transformed phenotype: rapid proliferation, loss of contact inhibition, and anchorage-independent growth. This transformation is dependent on eIF4E's ability to bind eIF4G, as co-expression of 4E-BP1 in these cells can partially reverse their malignant properties. Elevated eIF4E levels are detected in cancers of the breast, head, neck, bladder, colon, prostate, gastrointestinal tract and lung, Hodgkin's lymphomas, and neuroblastomas. In breast cancer patients, the risk of cancer recurrence and cancer-related death is correlated with the level of eIF4E overexpression. The other components of eIF4F are overexpressed in specific types of cancer: eIF4G in squamous cell lung carcinomas, and eIF4A in melanomas and primary hepatocellular carcinomas.
Loss of proper regulation of the eIF4E-eIF4G interaction plays an important role in the development of many cancers. The protein-protein interaction between eIF4E and eIF4G is an essential step in cap-dependent translation initiation. Because the translation of the mRNAs encoding most proteins involved in cellular growth and proliferation is highly cap-dependent, regulation of the level of complex formation between eIF4E and eIF4G plays an important role in the control of these processes. The interaction between these proteins is inhibited by the 4E binding proteins (4E-BPs), which compete with eIF4G for binding to the same surface on eIF4E. Phosphorylation of specific sites on 4E-BPs in response to growth and proliferation signals inhibits their ability to bind eIF4E.
The level of eIF4E/eIF4G complex formation also plays a role in the control of apoptosis. 4E-BP1 has been found to undergo a caspase cleavage of its C-terminus, which removes a motif necessary for it to undergo phosphorylation, leading to increased 4E-BP1 binding to eIF4E and inhibition of cap-dependent translation. This inhibition causes a shift in the levels of pro and anti apopoptic proteins to favor apoptosis. Experiments in cultured cells have shown that peptides containing the eIF4E recognition motif of eIF4G fused to a penetrating sequence can induce apoptosis.
In general, translation initiation is beneficial for inhibiting cellular proliferative disorders, whether cancerous or non-cancerous and translation initiation is an accepted target for cancer treatments. See Funda Meric and Kelly Hunt, Translation Initiation in Cancer: A Novel Target for Therapy, Molecular Cancer Therapeutics, Vol. 1, 971-979, September 2002; S. J. Watkins and C. J. Norbury, Translation Initiation and Its Deregulation During Tumorigenesis, British Journal of Cancer (2002) 86, 1023-1027; Igor Rosenwald, The Role of Translation in Neoplastic Transformation from a Pathologist's Point of View, Oncogene (2004) 23, 3230-3247; Igor Rosenwald, Songtao Wang, Lou Savas, Bruce Woda, James Pullman, Expression of Translation Initiation Factor eIF-2α is Increased in Benign and Malignant Melanocytic and Colonic Epithelial Neoplasms, Cancer, Vol. 98, No. 5, (2003); Songtao Wang, Igor Rosenwald, Michael Hutzler, German Pihan, Lou Savas, Jane-Jane Chen and Bruce Woda, Expression of the Eukaryotic Translation Initiation Factors 4E and 2α in Non-Hodgkin's Lymphomas, American Journal of Pathology, Vol. 155, 247-255 (1999); B. Bilanges and D. Stokoe, Mechanisms of Translational Deregulation in Human Tumors and Therapeutic Intervention Strategies, Oncogene (2007) 26, 5973-5990; Songtao Wang, Ricardo Lloyd, Michael Hutzler, Igor Rosenwald, Marjorie Safran, Nilima Patwardhan and Ashraf Khan, Expression of Eukaryotic Translation Initiation Factors 4E and 2α Correlates with the Progression of Thyroid Carcinoma, Thyroid, Vol. 11, No. 12 1101-1107 (2001).